结核分枝杆菌rmlB和rmlC基因是分枝杆菌生长相关基因

发布时间：2018-04-23 13:10

  本文选题：结核分枝杆菌 + dTDP-鼠李糖　； 参考：《大连医科大学》2005年博士论文

【摘要】：结核分枝杆菌(M．tuberculosis)是引起结核病的病原体。由于耐多药菌株的出现和HIV的协同作用，使得一度被控制的结核病的发病率在全球范围内显著回升，因此，寻找新一代抗结核药物已迫在眉睫。细胞壁是分枝杆菌赖以生存的结构基础，其核心结构由分枝菌酸、聚阿拉伯糖半乳糖及肽聚糖三种组分组成。分枝菌酸和聚阿拉伯糖半乳糖通过衔接双糖(L-鼠李糖-D-N-乙酰葡糖胺)共价连接到肽聚糖大分子上，衔接双糖尤其是鼠李糖可作为研发新一代抗结核药物的理想靶标。衔接双糖中鼠李糖的糖基供体是dTDP-鼠李糖，四种酶(Rm1A-D)参与了由底物葡萄糖-1-磷酸合成dTDP-鼠李糖的过程，，编码这四种酶的rm1A-D基因存在于结核分枝杆菌基因组中不同的位置，其中rm1B和rm1C同属于一个操纵子。尽管我们已发现在结核分枝杆菌中不存在dTDP-鼠李糖生物合成的补救代谢途径，但仍有必要从理论上证明编码结核分枝杆菌dTDP-鼠李糖合成酶系的rm1A-D基因为结核分枝杆菌的生长相关基因。 本论文的目的是用基因剔除方法证明结核分枝杆菌中编码Rm1B(dTDP-D-葡萄糖-4，6-脱氢酶)和Rm1C(dTDP-4-酮基-6-脱氧-D-葡萄糖3，5表异构酶)的rm1B和C基因是分枝杆菌生长过程中所必需的基因，这将为dTDP-鼠李糖作为研发抗结核新药的理想靶标提供更有力的证据。 利用与结核分枝杆菌有相同细胞壁结构，但生长快速且无致病性的耻垢分枝杆菌(Mycobacterium smegmatis，Sm)mc~2155菌株作为实验模型。用携带Sm rm1B：：Kan~R突变基因的条件复制性质粒转化mc~2155菌株，在42℃条件下筛选出mc~2155突变菌株M1(其基因组中同时存在Sm rm1B和Sm rm1B：：Kan~R)。用携带结核分枝杆菌M．tuberculosis，Tb)rm1B和rm1C基因的营救质粒转化mc~2155 M1突变菌株，Tb rm1B和Tb rm1C基因的表达产物可补偿mc~2155 M1突变菌株中突变的Sm rm1B以及突变的Sm rm1C基因的功能，在30℃条件下筛选出mc~2155 M2突变菌株(其基因组中只存在Sm rm1B：：Kan~R，即Sm rm1B基因剔除；同时Sm rm1B：：Kan~R下游的Sm rm1C基因发生框移突变)。分别在30℃和42℃条件下测定
[Abstract]:Mycobacterium tuberculosis M. tuberculosis is the pathogen of tuberculosis. Due to the emergence of multi-drug resistant strains and the synergistic effect of HIV, the incidence of once controlled tuberculosis has increased significantly in the world. Therefore, it is urgent to find a new generation of anti-tuberculosis drugs. The cell wall is the structural basis for the survival of Mycobacterium. Its core structure consists of three components: mycoic acid, polyarabinose galactose and peptidoglycan. Mycoic acid and polyarabinose galactose are covalently linked to peptidoglycan macromolecules by linking disaccharide L- rhamnose-D-N- acetyl glucosamine, which can be used as an ideal target for the development of a new generation of antituberculotic drugs. The glycosyl donor linking rhamnose in disaccharide is dTDP- rhamnose. Four enzymes Rm1A-D) are involved in the synthesis of dTDP- rhamnose from glucose-1-phosphate substrate. The rm1A-D gene encoding these four enzymes exists in different positions in the genome of Mycobacterium tuberculosis. Rm1B and rm1C belong to the same operator. Although we have found that there is no rescue metabolic pathway for dTDP- rhamnose biosynthesis in Mycobacterium tuberculosis, However, it is necessary to prove theoretically that the rm1A-D gene encoding the dTDP- rhamnose synthase system of Mycobacterium tuberculosis is related to the growth of Mycobacterium tuberculosis. The purpose of this paper is to prove that rm1B and C genes of Mycobacterium tuberculosis encode Rm1BP- dTDP-glucose-4nd6- dehydrogenase and Rm1CndTDP-4-keto-6-deoxy-D- glucose-3 isoisomerase) are essential genes in the growth of Mycobacterium tuberculosis. This will provide stronger evidence for dTDP- rhamnose as an ideal target for new anti-TB drugs. Mycobacterium smegmatissimus smmccf2155, which had the same cell wall structure as Mycobacterium tuberculosis, but grew rapidly and without pathogenicity, was used as the experimental model. Mc~2155 strain was transformed by conditional replicative plasmid carrying Sm rm1B::Kan~R mutant gene. Mc~2155 mutant strain M1 was screened at 42 鈩

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